Urinary complement factor D is increased in primary malignant hypertension: a single-center, cross-sectional study

Kidney injury is one of the detrimental consequences of primary malignant hypertension (pMHTN). There is a paucity of non-invasive biomarkers to enhance diagnosis and elucidate the underlying mechanisms. This study aims to explore urine protein biomarkers for pMHTN associated renal damage. In the discovery phase, urine samples were collected from 8 pMHTN, 19 disease controls (DCs), and 5 healthy controls (HCs). In-gel digestion combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) approach was used for identification of proteins associated with pMHTN. In the validation phase, the differentially expressed proteins were validated by ELISA assay in cohort with 10 pMHTN patients, 37 DCs, and 30 HCs. Compared to DCs and HCs, a specific band between 15 and 25 kDa was found in 7 out of 8 patients with pMHTN. Further LC–MS/MS analysis revealed 5 differentially expressed proteins. ELISA validation demonstrated that urinary complement factor D (CFD) was significantly up regulated in pMHTN. By receiver operating characteristic curve analysis, urinary CFD/Cr showed moderate potential in discriminating pMHTN from DCs (the area under curve: 0.822, 95% CI 0.618–0.962). Urinary CFD may be a potential biomarker for pMHTN with its elevation indicative of the activation of the alternative complement pathway in pMHTN.

cancer 11 , and colorectal carcinoma 12 .For certain conditions, urine may serve as a more sensitive and specific biomarker source than blood 13,14 .Proteomic analysis of urine has the potential to identify non-invasive biomarkers for pMHTN.
In the present study, we initially performed urinary sodium sodecy sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of patients with various diseases and observed distinct protein bands from pMHTN patients compared to the control groups.Further qualitative and quantitative analyses of the differential proteins were conducted using in-gel digestion followed by two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing specific urinary proteins associated with pMHTN.Subsequently, enzyme-linked immunosorbent assay (ELISA) validation in an independent cohort verified urinary complement factor D (CFD) as a noninvasive diagnostic biomarker for pMHTN.These findings contribute novel insights into the noninvasive diagnosis of pMHTN and complement-mediated pathogenesis of the disease.
Through SDS-PAGE analysis, we identified a distinct band between 15 and 25 kDa that was highly expressed in pMHTN patients (M1-3) compared to other renal diseases.This band may represent a potential protein biomarker for pMHTN (Fig. 1A).Further SDS-PAGE analysis involving pMHTN, non-renal DCs, and HCs revealed that this specific band was present in 4 out of 5 pMHTN patients (M4, 5, 6, 8), while it was absent in DCs and HCs (Fig. 1B).The gel images were cropped for clarity and the full-length gels are presented in Supplementary Materials (Supplementary Fig. 1 & 2).Lanes corresponding to additional replicates of the experiment and samples from patients with ambiguous diagnoses were omitted, but without affecting the conclusions.

LC-MS/MS identification of differential proteins
In the analysis of high-expression protein bands within 15-25 kD from three pMHTN patients (M1, M2, M3), in-gel digestion followed by MS identified 11 proteins.Compared to corresponding range bands in three HCs, a quantitative assessment using spectrum counting identified five differential proteins.The differential proteins identified include CFD, pancreatic ribonuclease, lithostathine-1-alpha, RBP4 and agrin (Table 2).

ELISA validation of differential proteins
During the validation phase, we recruited 10 pMHTN patients, 37 DCs, and 30 HCs (Table 3).The disease control group consisted of 5 benign hypertensive nephrosclerosis, 13 IgA nephropathy (IgAN), 6 LN, and 13 MN.Among the 10 pMHTN patients, one presented with microangiopathic hemolytic anemia and thrombocytopenia, suggestive of MHTN with systemic TMA.The remaining patients had normal levels of hemoglobin, platelets, and LDH (Supplementary Table 2).
We further validated the urinary levels of CFD/Cr, CAF/Cr, and RBP4/Cr using ELISA across three groups (Fig. 2).Amongst these urinary proteins, CFD/Cr exhibited significant differences, with the pMHTN group showing substantially higher values than both DC and HC groups (p < 0.001).In contrast, there were no differences observed in urinary RBP4/Cr and CAF/Cr between the pMHTN group and DC group (Fig. 2a-c).
To minimize bias arising from renal function and urinary protein levels, we employed the propensity score matching (PSM) to match the 24-hUP and eGFR between the pMHTN group and DC group.After matching, aside from age and blood pressure, there were no statistically significant differences in baseline data between the two groups (Table 3).After PSM analysis, the urinary CFD/Cr levels still revealed significant differences, whereas no statistical differences were found for RBP4/Cr and CAF/Cr (Fig. 2d-f).

Discussion
pMHTN is a renal emergency that can lead to deterioration of kidney function, and there is a lack of non-invasive biomarkers to improve diagnosis and unravel its pathophysiological mechanisms.In this study, we found significantly elevated levels of urinary CFD in biopsy-proven pMHTN patients through SDS-PAGE and LC-MS/ MS analysis.The validation analysis revealed that urinary CFD levels were markedly increased in the pMHTN group compared to those with IgAN, MN, LN, and benign hypertensive nephrosclerosis.These data suggest the potential of urinary CFD as a diagnostic biomarker for pMHTN and indicate the involvement of the alternative complement pathway activation in the pathogenesis of pMHTN.
Past research considered shear stress-induced endothelial damage as the main contributor to renal injury in MHTN.However, recent studies have indicated that complement activation, inflammation, and oxidative stress could also cause microvascular endothelial damage 15 .In particular, the activation of the alternative pathway (AP) may play a critical role in renal injury of pMHTN [16][17][18] .Timmermans et al. reported patients with primary severe hypertension accompanied by renal TMA who exhibited mutations in genes encoding for complement components.The activation of the complement system was evidenced by increased plasma levels of soluble C5b-9 and deposition of C4d, C3c, and C5b-9 on renal vascular and glomerular capillary walls, suggesting that severe hypertension could trigger complement activation, leading to the occurrence of TMA 17 .Additionally, serum samples collected at diagnosis from these patients induced abnormal C5b-9 formation on microvascular endothelium, reflecting active complement dysregulation 16 .However, these studies did not evaluate urinary AP-related complement levels.Zhang et al. assessed AP pathway-associated complements in the urine and plasm between pMHTN and healthy controls, and found significantly higher urinary CFD levels in pMHTN patients, aligning with our findings 18 .However, this study only compared pMHTN patients with a healthy control group, lacking an analysis against other kidney diseases, thus not assessing the potential diagnostic value of urinary CFD for CFD is a 24 kDa serine protease consisting of 228 amino acids, with a normal blood concentration of approximately 1-2 ug/mL 19 .CFD is involved in the initiation and regulation of the AP, acting as a critical rate-limiting enzyme for this pathway 19 .CFD is completely reabsorbed in the renal tubules following glomerular filtration and is rapidly degraded at the intracellular level.Kidneys regulate blood CFD concentration through the glomerular  www.nature.com/scientificreports/filtration rate 20 .Given that both serum and urine CFD levels are significantly elevated in patients with renal insufficiency 21,22 , we matched the eGFR of the pMHTN group with the DC group, and urinary CFD/Cr levels were approximately tenfold higher in the pMHTN group compared to DC group after PSM.The elevated urinary CFD levels in pMHTN may be related to systemic dysregulation of the AP or local complement activation within the kidneys.Nonetheless, there is a distinct possibility that hypertension may be provoked by complement abnormalities, which in turn aggravate hypertension and result in positive feedback dysregulation of the complement system 23 .Cavero et al. 24 provide additional persuasive evidence regarding the fundamental role of the complement system in hypertension-associated TMA.In their cohort of 55 atypical hemolytic uremic syndrome (aHUS) patients, 36 exhibited grade II or III hypertension while 19 showed grade III/IV retinopathy.Genetic complement abnormalities existed in 37% of the MHTN group.Antihypertensives were administered to all patients but only one exhibited hematological and renal responses.Eculizumab treatment resulted in renal and hematological  -c).Amongst three urinary proteins, CFD/Cr exhibited significant differences, with the pMHTN group showing substantially higher values than both disease control and health control groups (p < 0.001).In contrast, there were no differences observed in urinary RBP4/Cr and CAF/Cr between the pMHTN group and disease controls group.The expression of three differential proteins in the validation cohort before PSM (d-f).The urinary CFD/Cr levels still revealed significant differences, whereas no statistical differences were found for RBP4/Cr and CAF/ Cr. responses in 7 of 9 MHTN patients.In our two pMHTN cohorts, only one patient had hypertension-associated TMA.It remains unclear whether complement dysregulation is present in our pMHTN patients.
Several small-molecule CFD inhibitors have entered clinical trials for treating diseases mediated by the AP, such as C3 glomerulopathy, and aHUS 19 .Although effective blood pressure management in MHTN can lead to favorable renal and overall outcomes, a subset of patients continued disease progression 3,5 .Our study, alongside existing data, suggested CFD inhibitors may serve as a targeted treatment for pMHTN, especially when complicated by TMA.Future research should investigate the dysregulation of complement activity in pMHTN as a potential therapeutic direction.
Through LC-MS/MS analysis, we also identified other two differential proteins: Lithostathine-1-alpha and Pancreatic ribonuclease.Lithostathine-1-alpha is predominantly secreted by the exocrine pancreas and may play a role in pancreatic stone formation and the pathogenesis of diabetes mellitus 25,26 .Pancreatic ribonuclease, which participates in RNA cleavage, is a pyrimidine-specific endoribonuclease highly expressed in pancreatic tissue 27 .To our knowledge, there are no reports linking these two proteins with kidney diseases.In an independent cohort, we validated RBP4 and the C-Terminal Fragment of Agrin.Urinary RBP4 is a biomarker for proximal tubular renal disease 28 .Agrin is a type of heparan sulfate proteoglycan that constitutes an essential part of the glomerular basement membrane and extracellular matrix 29 .With a molecular weight of 215 kDa, agrin does not conform to the molecular weight range of differential bands, however, the CAF is a product of Agrin cleavage by proteases, specifically with a molecular weight of 22 kDa.CAF is filtered by the glomeruli and reabsorbed in the renal tubules 30 .Our results indicate no significant differences in the levels of urine RBP4 and CAF between the pMHTN group and the DC group, suggesting that the diagnostic specificity of these two proteins for pMHTN is limited.
One limitation of our study is the small number of patients included, and its cross-sectional design, which precludes determining a causal relationship between CFD and pMHTN.Moreover, our study did not assess the urinary CFD levels in patients with secondary MHTN.There is also a lack of validation of CFD deposition in renal tissue.Thirdly, the pMHTN patients were younger than the control groups in the validation cohort.The potential influence of age on urinary CFD levels cannot be ruled out.Future large-scale longitudinal cohort studies are warranted to verify the role of CFD in pMHTN and to explore the relationship between urinary CFD levels and disease progression in pMHTN.

Conclusion
We report for the first time that the levels of urinary CFD/Cr are significantly elevated in biopsy-proven pMHTN patients and can serve as a potential diagnostic biomarker for pMHTN.There is a need for prospective studies with larger samples to validate this finding and explore the role of CFD in the pathogenesis and progression of pMHTN.Since pMHTN may involve the activation of AP, CFD inhibitors may be a potential therapeutic option for some patients with pMHTN.

Participants and study design
The study was conducted in two phases.In the first phase, we selected all biopsy-proven pMHTN patients from Peking Union Medical College Hospital and had complete clinical and pathological data between July 2013 and July 2014.Concurrently, hospitalized patients with other diseases during the same period were randomly chosen as the DCs), alongside healthy individuals as HCs.Urinary SDS-PAGE analysis revealed differential bands, and the proteins within these bands were subsequently digested with in-gel proteases and identified using two-dimensional LC-MS/MS.In the second phase, we prospectively recruited all patients with biopsy-proven pMHTN at our hospital between June 2017 and September 2018.As the DCs, patients with other renal diseases diagnosed by renal biopsy from June 2018 to September 2018 were included.The differential proteins identified by LC-MS/MS were validated using the ELISA method (Fig. 4).
The diagnosis of pMHTN must concurrently meet the following criteria: (1) Severe hypertension: diastolic blood pressure > 120 mmHg and/or systolic blood pressure > 180 mmHg on multiple measurements; (2) Fundoscopic examination: bilateral retinal arteriolar blot-or flame-shaped hemorrhages, or "cotton wool" exudates, possibly accompanied by papilledema (Keith-Wagener classification of Grade III or IV); (3) Renal pathology: characteristic histopathological changes of malignant nephrosclerosis, including intimal thickening and fibrosis with luminal narrowing of the interlobular and afferent arterioles, concentric myointimal proliferation ('onion skin' lesions), possibly with fibrinoid necrosis or thrombosis.Pathological examination of renal tissues included light microscopy (Hematoxylin & Eosin, Periodic Acid-Schiff, Periodic Acid-Silver Methenamine, and Masson's Trichrome staining), electron microscopy, and immunofluorescence for IgG, IgM, IgA, C3, C4, C1q, fibrinogen, HBsAg, and HBcAg.The renal biopsy specimens were independently assessed by two nephropathologists.Exclusion criteria included secondary hypertension, such as renal parenchymal and renovascular hypertension and endocrine-related hypertension.Endocrine-related secondary hypertension was investigated with measurements of plasma renin/angiotensin II/aldosterone levels in supine and upright positions, thyroid hormones, serum ACTH, 24-h urinary catecholamines, and 24-h urinary free cortisol levels.
We collected various clinical and laboratory parameters, including age, gender, body mass index, blood pressure, hemoglobin levels, platelet count, complement C3 and C4 levels, lactate dehydrogenase (LDH), 24-h urinary protein (24hUP), serum creatinine, estimated glomerular filtration rate (eGFR), blood glucose, lipid profiles, and types of antihypertensive medications administered.The eGFR was assessed using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

Urine collection and preservation
Midstream urine samples (30 mL) were collected from patients on the morning of renal biopsy.The samples underwent centrifugation at 3000 rpm for 10 min, followed by filtration through nitrocellulose membranes to adsorb urinary proteins.The membranes were dried and stored at − 80 °C in vacuum-sealed bags.

Urine SDS-PAGE and in-gel digestion
Urine proteins preserved on nitrocellulose membranes were eluted by heating and protein concentrations were measured using the Bradford method.The eluted proteins were mixed with PAGE sample buffer (50 mM Tris-HCl, pH 6.8 with 50 mM DTT, 0.5% SDS and 10% glycerol), heated at 95 °C for 5 min, and then separated by SDS-PAGE using 12% acrylamide gels.After electrophoresis, the differential bands between 15 and 25 kD

Figure 2 .
Figure 2. The expression of three differential proteins in the validation cohort before PSM (a-c).Amongst three urinary proteins, CFD/Cr exhibited significant differences, with the pMHTN group showing substantially higher values than both disease control and health control groups (p < 0.001).In contrast, there were no differences observed in urinary RBP4/Cr and CAF/Cr between the pMHTN group and disease controls group.The expression of three differential proteins in the validation cohort before PSM (d-f).The urinary CFD/Cr levels still revealed significant differences, whereas no statistical differences were found for RBP4/Cr and CAF/ Cr.

Table 1 .
Clinical features and laboratory findings of patients in the discovery phase.M male, F female, pMHTN primary malignant hypertension, MHTN malignant hypertension, MN membranous nephropathy, LN lupus nephropathy, IgAN IgA nephropathy, HTN, hypertension, BD Behcet's disease, DM diabetes mellitus, N.D. not done, N none, CCB calcium channel blocker, ARB angiotensin II receptor blocker; ACEI angiotensinconverting enzyme inhibitor.Hypertension-related retinopathy classification according to the classification of Keith-Wagener-Barker. *Negative proteinuria by urinalysis.

Table 2 .
The details of the differentially expressed proteins in pMHTN patients.MW molecular weight, HC healthy control, pMHTN primary malignant hypertension, FC fold change.The p value represents the statistical difference in the mass spectrum numbers between pMHTN and HCs.

Table 3 .
Clinical characteristics of participants in the validation cohort before and after PSM.PSM propensity score matching, pMHTN primary malignant hypertension, DC disease control, HC healthy control, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, Scr serum creatinine, eGFR estimated glomerular filtration rate, TG triglyceride, 24hUP 24-h urinary protein.*p < 0.05 pMHTN vs DC, # p < 0.05 pMHTN vs HC.Significant values are in bold.